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5 - Estimation of starch synthase

5 - Estimation of starch synthase

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100



CHAPTER 12  Carbohydrates and related enzymes



6. The pellet finally obtained is resuspended in 1 mL of the same buffer to obtain

globule-bound starch synthase activity (George et al., 1994).

Estimation:

1. In a test tube, take 0.1 mL of the enzyme extract (supernatant) and add

0.25 mL of reaction mixture containing (25 mM Tris-HCl buffer, pH 8.5,

0.2 mM EDTA, 2.5 mM glutathione, 5 mM KC1, 0.5 mg glycogen, and

0.25 mM ADPG).

2. The reaction is started by adding the enzyme extract and stopped later by

immersing the tube in a boiling water bath for 1 min.

3. The tubes are cooled immediately in a water bath at room temperature.

4. A blank is run without the substrate ADPG and amount of ADP liberated is

then determined by pyruvate kinase method (Leloir and Goldenburg, 1960).

5. To estimate the ADP formed in the above reaction, 0.025 mL of phosphoenol

pyruvate (0.01 M solutions in 0.4 M KC1) and 0.025 mL of pyruvate kinase

(8.4 U freshly diluted in 0.1 M MgSO4) are added and incubated for 15 min

at 37°C.

6. At the end of the incubation period, 0.15 mL of dinitrophenyl hydrazine (0.1%

in 2 N-HC1) is added.

7. After 5 min, 0.2 mL of 10 N NaOH and 1.1 mL of 95% ethanol are added.

8. The samples are mixed and centrifuged and the OD of the supernatant is

measured at 520 nm.

9. Results are calculated from a standard curve drawn by using different

concentrations (10–100 nmol) of pyruvate.

10. The enzyme activity is expressed on fr.wt. or dry weight basis.



12.6  ESTIMATION OF INVERTASES

1. Invertases are prevalent in storage tissues and can catalyze the hydrolysis of

sucrose to glucose and fructose.

2. This reaction is termed “inversion reaction” because during the reaction the

optical rotation shifts from +66.5°C to –28°C.

3. Invertase is also called “sucrose.”

4. Since the hydrolysis is not reversible, sucrase cannot catalyze the synthesis of

sucrose.

Principle: The enzyme sucrose phosphate synthase catalyzes the synthesis of sucrose

and the enzymes invertase and sucrose synthetase both catalyze the hydrolysis of

sucrose, the later producing the nucleotide diphosphate derivatives of glucose for

subsequent biosynthetic reactions.

The enzyme invertase can be quantitatively assayed following a method as described by Tang et al. (1999).



12.6 Estimation of invertases



Chemicals required:

• Sodium acetate (C2H3O2Na.3H2O)

• Acetic acid

• b-Mercaptoethanol

• Lysine

• Ethylene diamine tetra acetic acid (EDTA)

• Phenylmethane sulfonyl fluoride.

• Sodium chloride (NaCl)

• Sucrose

• Citric acid

• Sodium carbonate (Na2CO3)

• Rochelle salt–sodium potassium tartarate

• Copper sulfate

• Sodium bicarbonate (NaHCO3)

• Sodium sulfate (Na2SO4)

Preparation of reagents:

Sodium acetate buffer (25 mM; pH 5.0)

A: 0.5% b-mercaptoethanol (500 mL in 100 mL) + 25 mM solution of acetic

acid (1.444 mL in 1000 mL D.D H2O water).

B: 25 mM solution of sodium acetate (C2H3O2Na·3H2O): 0.3402 g of sodium

acetate in 100 mL of D.D H2O water).

14.8 mL of A + 35.2 mL of B, diluted to a total of 100 mL. Adjust the pH (5.0)

• Lysine (10 mM)

• EDTA (1 mM) : 37.2 mg in 100 mL of D.D.H2O

• 0.1 mM phenyl methane sulfonyl fluoride.

• NaCl (1 M): 5.844 g in 100 mL of D.D.H2O water.

Cell wall invertase:

• Sucrose (50 mM)

• Citric acid (13.5 mM): 259.36 mg in 100 mL of D.D.H2O.

• Disodium phosphate, 26.5 mM (pH 4.6): 376.19 mg in 100 mL D.D.H2O.

Soluble invertase:

• Citric acid (10.5 mM): 201.72 mg in 100 mL of D.D.H2O

• Disodium phosphate (29.0 mM) (pH 5.4): 411.68 mg in 100 mL of D.D.H2O

• Alkaline copper carbonate tartrate reagent according to Somogyi (1952).

Extraction:

1. Leaves (2 g) of mature plants are homogenized in 10 mL of ice-cold sodium

acetate buffer (25 mM; pH 5.0) containing 0.5% b-mercaptoethanol, 10 mM

lysine, and 1 mM EDTA and 0.1 mM phenylmethane sulfonyl fluoride.

2. The homogenate is filtered through a 4-layered muslin cloth.

3. It is centrifuged at 10,000× g for 30 min at 0.4°C.



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CHAPTER 12  Carbohydrates and related enzymes



4. The supernatant is used for the determination of acid soluble invertase activity.

5. The pellets are washed extensively with ice-cold water and cell wall proteins are

extracted with 5 mL of 1 M NaCl overnight at 4°C.

Estimation:

Cell wall invertase: The assay mixture consists of 50 mM sucrose, 13.5 mM

citric acid, 26.5 mM disodium phosphate (pH 4.6), and pellet extract all in a

total volume of 3.0 mL.

Soluble invertase: Constituents of assay mixture are 50 mM sucrose, 10.5 mM

citric acid, 29.0 mM disodium phosphate (pH 5.4), and the supernatant all in a

total volume of 3.0 mL.

The reaction mixture is incubated at 37°C for 15–20 min.

The reaction is stopped with alkaline copper reagent and the liberated reducing

sugars are measured following the method of Somogyi (1952).



CHAPTER



Nitrogen compounds and

related enzymes



13



Plants take up nitrogen from soil either in the form of nitrate or ammonia. Nitrate

taken by plants is reduced by nitrate reductase and nitrite reductase enzymes. They

catalyze step-wise reduction of nitrate to nitrite and nitrite to ammonia. Ammonia

occupies the key position in nitrogen metabolism for the synthesis of organic nitrogen. Glutamic acid dehyrogenase (GDH), glutamate synthase (GOGAT), and glutamine synthatase (GS) are important enzymes that are involved in the conversion of

ammonia into glutamic acid, a primary amino acid. The a-amino group of several

amino acids is formed as a result of amino transferase enzymes. This process is of

great significance in amino acid metabolism. Protocols for estimation of all the important enzymes associated with nitrogen metabolism as well as total nitrogen and

free amino acids are given in this chapter.



13.1  TOTAL NITROGEN

13.1.1  KJELDHAL METHOD FOR QUANTIFYING LEAF

NITROGEN CONTENT

Measurement of leaf nitrogen can be made using the Kjeldhal method. The ­Kjeldhal

method for quantifying leaf nitrogen status involves digesting a dried and finely powered sample in a medium containing a strong acid such as sulfuric acid to produce

ammonium sulfate, followed by liberation of ammonia by adding a strong alkali–

sodium hydroxide. The ammonia is then captured using boric acid and the exact

amount of nitrogen can be determined by titrating the excess acid with sodium carbonate. This method is invariably followed in this laboratory for computing specific

leaf nitrogen in crop genotypes. Although the Kjeldhal method is fairly accurate, it is

quite cumbersome and time-consuming.

Principle:

A known weight of the powdered sample is treated with sulfuric acid so as to

oxidize the organic matter and bring the mineral elements into solution.

Chemicals required:

Sulfuric acid

Potassium sulfate

Copper sulfate (CuSO4·5H2O)

Boric acid

Phenotyping Crop Plants for Physiological and Biochemical Traits. http://dx.doi.org/10.1016/B978-0-12-804073-7.00013-2

Copyright © 2016 BSP Books Pvt. Ltd. Published by Elsevier Inc. All rights reserved.



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CHAPTER 13  Nitrogen compounds and related enzymes



Bromocresol green

Methyl red double indicator

Sodium hydroxide



13.1.2  PREPARATION OF REAGENTS

Receiver solution: Receiver solution of 1 L was prepared by adding 40 g of boric

acid, 10 mL of bromocresol green, 7 mL of methyl red solution into a conical flask

and making up to 1 L with distilled water.

40% sodium hydroxide: 40 g of sodium hydroxide in 100 mL of distilled water

N sulfuric acid: 2.7 mL of sulfuric acid in 1 L of distilled water

Digestion: Transfer 1 g of powdered sample into digestion tube. Add 0.8 g of copper

sulfate and 7 g of potassium sulfate to the digestion tube. Concentrated sulfuric acid

(12 mL) was added slowly to the digestion tube. The digestion tube was kept on a

digestion plate and heat at 420oC for about 30–45 min. Continue to digest until the

sample turns to blue/green color.

Distillation: Remove the digestion tubes and cool the tubes for 10–20 min. Add

75 mL of deionised water to digestion tube and keep it in distillation unit. Add 25 mL

of receiver solution into 250-mL conical flask and place it into distillation unit.

Raise the platform so that the distillate outlet is submerged in the receiver solution.

­Dispense 50 mL of 40% sodium hydroxide into the digestion tube. Open the steam

valve and distill approximately for 4 min. The receiver solution in the distillation unit

will turn to green color indicating the presence of alkali ammonia. Take out the flask

having receiver solution and titrate against 0.1 N sulfuric acid.

Calculation:

Percent nitrogen (% N) =



(T − B) × N × 14.007 × 100

Weigth of sample in mg



where

T = titre value

B = blank value

N = normality of sulfuric acid (0.1 N)



13.1.3  PROTEIN PERCENT CAN BE DETERMINED INDIRECTLY USING

THE FOLLOWING FORMULA

Percent protein = 6.25 × % N (A.O.A.C,1960)



Other methods: More sophisticated instruments such as elemental analyzers provide

a high-throughput measurement option for accurate determination of leaf nitrogen. In

this technique, a small quantity of the homogenized sample is completely combusted

in a temperature-controlled reactor filled with appropriate catalysts. In the presence

of excess oxygen for combustion, the nitrogen in the sample is oxidized to produce



13.2 Total free amino acids



nitrogen oxides. These nitrogen oxides are subsequently reduced to form nitrogen gas.

The quantity of nitrogen gas is determined by a temperature conductivity detector.



13.2  TOTAL FREE AMINO ACIDS

The amino acids are the organic compounds that form the basic building blocks of

proteins. The common feature of the structure of amino acids is having a minimum

of two ionizable groups: the acidic carboxyl (-COOH) and the basic amino (-NH2)

groups on the same carbon atom called a-carbon atom. The amino acids that also exist in the free form and are not bound to proteins are known as free amino acids. They

are mostly water soluble in nature. Very often in plant during disease conditions, the

free amino acids composition exhibits a change and hence, the measurement of

the total free amino acids gives the physiological and health status of the plants.

Principle: Ninhydrin, a powerful oxidizing agent, decarboxylates the alpha-amino

acids and carboxylates to give an intensely colored bluish purple product that is

colorimetrically measured at 570 nm (Moore and Stein, 1948; Misra et al., 1975;

­Theymoli Balasubramanian and Sadasivam, 1987).

Ninhydrin + α -amino acid → Hydrindantin + decarboxylated Aminoacid

+ carbon dioxide + Ammonia.

Hydrindantin + Ninhydrin + Ammonia → Purple colored product + Water



Chemicals required:

• Ninhydrin

• Stannous chloride

• Citric acid

• Sodium citrate

• Methyl cellosolve

• N-propanol

• Ethanol

• Leucine

Preparation of reagents:

• Ninhydrin: dissolve 0.4 g stannous chloride (SnCl2·2H2O) in 250 mL of 0.2 M

citrate buffer (pH 5.0). Add this solution to 10 g of ninhydrin in 250 mL of

methyl cellosolve (2-methoxyethanol). Prepare freshly and store in brown bottle

(carcinogenic).

• 0.2 M citrate buffer (pH 5.0)

• Solution A: 0.2 M citric acid solution

• Solution B: 0.2 M sodium citrate (C6H5O7 Na3·2H2O) solution

Add 20.5 mL of solution A and 29.5 mL of solution B diluted to a total of 100 mL

with distilled water (pH 5.0).

Diluent solvent: mix equal volumes of water and n-propanol, and use.



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CHAPTER 13  Nitrogen compounds and related enzymes



Extraction:

1. Weigh 500 mg of the plant sample and grind it in a pestle and mortar with

5–10 mL of 80% ethanol.

2. Filter or centrifuge. Save the filtrate or the supernatant.

3. Repeat the extraction twice with residue and pool all the supernatants.

4. Reduce the volume if needed by evaporation and use the extract for the

quantitative estimation of total free amino acids.

5. If the tissue is tough, use boiling 80% ethanol for extraction.

Estimation:

1. To 0.1 mL of extract add 1 mL of ninhydrin solution and mix.

2. Make up the volume to 2 mL with distilled water.

3. Heat the tube in a boiling water bath for 20 min.

4. Add 5 mL of the diluents and mix the contents.

5. After 15 min, read intensity of purple color against a reagent blank in a

spectrophometer using photometric method at 570 nm.

6. The color is stable for 1 h. Prepare the reagent blank as above by taking

0.1 mL of 80% ethanol instead of the extract.

7. Dissolve 50 mg leucine in 50 mL of distilled water in a volumetric flask.

8. Take 10 mL of this stock standard and dilute to 100 mL with distilled water in

another volumetric flask for working standard solution.

9. A series of volumes from 0.1 to 1.0 mL of this standard solution gives a

concentration range 10–100 mg.

10. Then proceed as that of the sample and read the color.

Calculation: Draw a standard curve using absorbance versus concentration. Find out

the concentration of the free amino acids in the sample using standard regression

equation and express as mg per g fr.wt.



13.3  NITRATE REDUCTASE

The assimilatory reduction of nitrate by plant is a fundamental biological process in

which a highly oxidized form of inorganic nitrogen is reduced to nitrite and then to

ammonia.

NO3− + AH 2 → NO 2− + A + H 2 O



The nitrate reducing system consists of nitrate reductase and nitrite reductase

which catalyze stepwise reduction of nitrate to nitrite and then to ammonia. The

NADH-dependent NR is most prevalent in plants.

Principle: Nitrate reductase (NR) is capable of utilizing the reduced form of

pyridine nucleotides, flavins, or benzyl viologen as electron donors for reduction

of nitrate to nitrite. Here, NR activity in plants can be measured by following the

oxidation of NAD (P) H at 340 nm. However, NR activity is commonly measured by

spectrophometric determination of nitrite produced (Klepper et al., 1971).



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